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Isolation and Screening of Microorganisms
Nyoman S. Antara, Ph.D.Industrial Microbiology
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Isolasi Mikroorganisme
Mikroba di alam selalu tercampur Isolasi mikroba untuk produk yang spesifik Sumber mikroba Jenis Mikroba Produk spesifik:
– Metabolite products– Enzyme– Transformation compounds– Biomass
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Isolasi Bakteri
Memisahkan bakteri dari campuran mikroba
Kultur (spesies) tunggal untuk dipelajari lebih lanjut
Dua metode konvensional isolasi bakteri:– Metode cawan gores– Metode cawan tuang
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Cawan Gores
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Cawan Tuang
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Isolation of Lactic Acid Bacteria from Urutan
GYP medium (per L)
Glucose 10 gYeast extraxt 10 gPeptone 5 gBeef extract 2 gNa-acetate 3H2O 2 gTween 5% sol. 10 mlSalt sol.* 5 mlBromocresol purple 60 mgAgar 12 gCaCO3 5 g
pH 6.8*each ml contains: MgSO4 7H2O, 40 mg; MnSO4.4H2O, 2 mg; FeSO4.7H2O, 7 mg; NaCl, 2 mg
GYP medium
Urutan
Day 1
Day 2
Day 3
Day 5
IsolationFrom day 1 2 3 5 Total
Number of isolates 19 22 19 11 71
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Phenotypic characterization
Gram staining and catalase test
Cell morphologyGas production from glucoseUtilization of sugarsGrowth at various temperatures
13 representative isolates:
Stereoisomers of lactic acid
Sequence of 16S rDNA
71 isolates
Rod: 9 groupsCocci: 4 groups
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Penapisan/Screening
Seleksi mikroba tertentu Uji produksi Uji produktivitas Seleksi gen Rekayasa genetik
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Isolasi Actinomycetes
Actinomycetes : anggota family dari Actinomycetales Dieksploitasi untuk produksi antibiotik, enzim, dan
senyawa komersial lainnya Hampir 5000 paten sudah dihasilkan dari klas
actinomycetes Streptomyces merupakan genus yang paling banyak
dieksploitasi Actinomycetes dapat ditemukan di mana-mana,
tanah merupakan reservoar paling besar, juga diisolasi dari sedimen lumpur danau, sungai, dan juga dari fresh water dan marine environment
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Selective Pretreatment
Pretreatment Selection forDry heat, 120oC for 1 h Micromonospora,
Microbispora, and Streptosporangium
Moist heat, 45oC for 2 h Rhodococcus
Moist heat, 50oC for 10 min Elimination of spreading bacterial colonies on soil plate
Moist heat, 70oC for 30 min Micromonospora
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Selective Media
Actinomycetes dapat survive dan tumbuh pada media dengan nutrisi sangat rendah
Media dengan carbon-to-nitrogen ratio yang tinggi
Kandungan sumber karbon dan nitrogen yang kompleks mengurangi tumbuhnya bakteri
Ke dalam media harus ditambahkan cycloheximide (50 ug/ml) dan nystatin (50 ug/ml) untuk mencegah tumbuhnya fungi
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Incubation Condition
Suhu inkubasi 25 – 30oC Streptomyces diinkubasi selama 7 – 14 hari Micromonospora diinkubasi selama 14 – 21
hari Genera lain perlu tambahan waktu inkubasi 3 – 5 minggu
Actinomycetes termofilik diinkubasi pada suhu 45 – 50oC, selama 2 – 5 hari, cawan di cek setiap hari
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Preservation of Isolates
Isolate Actinomycetes jangan disimpan dengan agar miring
Lyofilisasi digunakan untuk preservasi dalam jangka waktu lama
Sering digunakan cryoprotective: susu skim, beef serum, 12% sucrose broth steril
Pembekuan juga bisa digunakan untuk mengawetkan Actinomycetes
Viabilitas terbaik bila disimpan pada suhu -70oC Penggunaan suhu -20 – 50oC dapat bertahan 1 – 5
tahun
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Isolation of Filamentous Fungi
Materials Collecting Samples Media Basic Techniques Purifying Cultures Maintaining Cultures Identification
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Filamentous Fungi
Eucaryotic organism Occupy a wide range of habitats Oriental foods, soy sauce and tempe, and European
cheeses Six general catagories of fungal products:
– Organic acids– Pharmaceuticals– Enzymes– Insecticides– Polysaccharides– biomass
Citric acid produced by A. niger